A liquid handling platform for standardised quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterising emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression (CFE) systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimising the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC ₅₀ ) of gentamicin by over 150-fold, while arr-3 increased the IC ₅₀ of rifampicin by approximately 20-fold compared to controls. Future work could extend this platform to characterise novel ARGs identified through genomic surveillance or rapidly profile activity of new or derivative antibiotics.